Methods and compositions for the detection of chromosomal aberrations

ABSTRACT

This invention relates generally to methods and compositions for direct detection of specific nucleic acid flanking sequences associated with structural chromosomal aberration breakpoints, by forming hybrids between the sequences and genetic probes, and detecting the probes. In particular aspects, the invention concerns detection of nucleic acid sequences in situ in chromosomes, and more specifically in cells, including interphase cells. Compositions of probes useful for detecting chromosomal translocations, in particular those associated with human leukemias, are also disclosed. An aspect of the invention is labelled probes that, when juxtaposed by formation of an aberration, are distinguishable and provide a pattern different from that of normal cells.

This is a continuation of application Ser. No. 07/784,222 filed Oct. 28,1991, now issued U.S. Pat. Ser. No. 6,025,126.

The government may have certain rights in this invention pursuant toresearch funding provided by the National Institutes of Health, NIHR29-CA44700.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods and compositions for directdetection of specific nucleic acid sequences associated with flankingregions of chromosomal aberration breakpoints, by forming hybridsbetween the sequences and genetic probes, and detecting the probes. Inparticular aspects, the invention concerns detection of nucleic acidsequences in situ in chromosomes, and more specifically in cells,including interphase cells. Compositions of probes useful for detectingchromosomal translocations, in particular those associated with humanleukemias, are also disclosed.

2. Description of the Related Art

Substantial proportions of human diseases and malformations trace theiretiology, at least in part, to genetic factors. Some of these factorsare present in the zygote, others occur later as somatic cells form.Detection of genetic factors associated with particular diseases ormalformations provides a means for diagnosis and treatment. For someconditions, early detection may allow prevention or amelioration of thedevastating courses of diseases.

One class of genetic factors are chromosomal aberrations, that is,deviations in the expected numbers and structure of chromosomes for aparticular species, and for particular cell types within a species.These may be constitutive i.e. present in the zygote, or inducedpost-zygotically in somatic (non-germinal cells) leading to mosaicism,that is a condition where both normal and abnormal cells are present.Chromosomes are the microscopically visible entities that are composedof the genetic material and, in higher organisms such as man, proteinsand RNA. The study of chromosomes is called “cytogenetics”.

There are several classes of structural aberrations that may involveautosomes or sex chromosomes or both. These aberrations are detected bynoting changes in chromosome morphology (band patterns). The bandpatterns may be only changed in one chromosome (intrachromosomal) or inmore than one chromosome (interchromosomal). Normal phenotypes may beassociated with these rearrangements if the amount of genetic materialhas not been altered, but physical or mental anomalies are expected ifthere is gain or loss of genetic material. Simple deletions(deficiencies) refer to loss of part of a chromosome. Duplication refersto addition of material to chromosomes. Duplication and deficiency ofgenetic material can be produced by simple breakage of chromosomes, byerrors during DNA synthesis, or as a consequence of segregation of otherrearrangements into gametes.

Translocations are interchromosomal rearrangements effected by breakageand transfer of part of chromosomes to different locations. Inreciprocal translocations, pieces of chromosomes are exchanged betweentwo or more chromosomes. Generally, the exchanges of interest arebetween nonhomologues. If all the original genetic material appears tobe preserved, this condition is referred to as balanced. Unbalancedforms have duplications or deficiencies of genetic material associatedwith the exchange; that is, something has been gained or lost “in theshuffle.”

One of the most exciting associations between chromosomal aberrationsand human disease, is that between chromosomal aberrations and cancer.These aberrations are generally not constitutive, i.e., present in thezygote, therefore are not present in all cells—only the abnormal ones. Amosaic condition is said to exist. For example, the Philadelphia (Ph¹)chromosome is an important cytogenetic finding in chronic myelogenousleukemia (CML) and acute lymphoblastic leukemia (ALL). This chromosomewas originally identified as a chromosome sized slightly smaller than a“G-group” chromosome. It was believed to be a deleted chromosome untildetection of a reciprocal translocation between chromosomes No. 9 and 22was reported by Rowley. A reciprocal translocation is one caused bybreakage of at least two chromosomes and reunion of the broken piece innew locations.(FIG. 1) This aberration was first found to be associatedwith CML, but is now known to have prognostic and diagnostic value formany hematopoietic malignancies, e.g. ALL.

It is not only the translocation per se that is of clinical interest,but rather the resulting fusion of the proto-oncogene abl from the longarm of chromosome 9 with the bcr gene of chromosome 22, a consistentfinding in CML. This genetic change leads to formation of a bcr-abltranscript that is translated to form a 210 kD protein present invirtually all cases of CML. This fusion can be detected by Southernanalysis for bcr rearrangements or by in vitro amplification (PCR) of acomplementary DNA (cDNA) transcript copied from CML mRNA. Inapproximately 95% of cases, the fusion gene results from a reciprocaltranslocation involving chromosomes 9 and 22, producing acytogenetically distinct small acrocentric chromosome called Ph¹. In theremaining cases the genetic rearrangement is more complex, and theinvolvement of the bcr and abl regions of chromosomes 9 and 22 may notbe apparent during analysis of banded metaphase chromosomes. Southernblots, PCR, and metaphase chromosome banding analysis providecomplementary, but incomplete, information on CML. They do not permit agenetic analysis on a cell by cell basis in a format in which theresults can be related to cell phenotype as judged by morphology orother markers. Thus, assessment of the distribution of the CML genotypeamong cells of different lineage and maturity has not been possible.

As an example of the prognostic value of chromosomal aberrations, inadult ALL, the Ph¹ chromosome is present in up to one-third of cases,and is associated with a high relapse rate and short survival. Inpediatric ALL it is much less common, but it remains one of the fewchromosomal abnormalities that continues to carry a poor prognosis inspite of newer, more intensive approaches to treatment. The accuratedetection of the Ph¹ is thus an important part of the diagnosticevaluation of patients with ALL.

Unfortunately, the cytogenetic diagnosis of the Ph¹ chromosome in ALLhas been limited. Cytogenetic analysis has a high failure rate in thisdisease, compared to other acute leukemias or to CML. Fewer than 70% ofcases have adequately banded chromosomes at metaphases in most reports.“Banding” is a morphological pattern revealed by treating chromosomes toreveal horizontal stripes which vary in width and staining intensity andare characteristic of specific chromosomal regions. As an alternative tocytogenetic analysis, recently, newer methods of chromosomal in situhybridization with non-isotopically labelled genetic probes haveimproved and extended the capabilities of cytogenetics. One of thesemethods is fluorescence in situ hybridization (FISH). In this method,probes are labelled with fluorescent signals that are detectable,generally by microscopic viewing of colors. Probes are nucleic acidsequences which bind to matching (homologous) sequences, e.g. onchromosomes. Although based on cytogenetic diagnosis, FISH may beperformed on interphase cells as well as on metaphases, and may beapplied directly to cells from either the peripheral blood or bonemarrow without the need for banded karyotypes. The diagnostic utility ofFISH with repetitive, centromeric probes in cases of leukemia has beendemonstrated in previous studies.

FISH on interphase cells has proven to be a useful method for diagnosisand clinical management in hematologic diseases. However, much of thisexperience has concentrated on detecting numerical chromosomalabnormalities (single chromosome loss or gain), making use ofchromosome-specific alpha satellite probes, which are highly-repetitive,unique sequences that occur within or near the centromere ofchromosomes. The centromere is a constriction most readily visible atmetaphase of cell division, which occurs at a characteristic location oneach chromosome. The development of competitive hybridization methods toeliminate the signal from Alu-type repeats, and improvements in opticsand reagents, have also made it possible to visualize single-copygenomic clones by FISH. However, the use of genomic clones is moredifficult than the use of alpha satellite probes, because of lowersignal intensity and high background. These difficulties would be offsetif use of genomic clones produced improvements in disease assayspecificity and were more flexible. Genomic clones are those thatcontain repeated sequences and non-coding sequences, that is DNA as itexists in the chromosome.

Some of the background for the present invention is as follows: singlestranded synthetic DNA was developed with multiple sites areincorporated where fragments may be used as probes. (Stephensen, U.S.Pat. No. 4,681,840). Oncogenes are genes whose products have the abilityto transform eukaryotic cells so that they grow in a manner analogous totumor cells. Probes and methods for detecting chromosomal translocationsare disclosed in EPO 181 635 (Groffen et al.)

Pinkel et al. (1986, 1988) and Gray et al. (1990) relatefluorescent-labeled probes for the cytogenetic analysis of chromosomes,and in situ hybridization of chromosomes at metaphase and interphasewith whole chromosome-specific DNA.

In situ hybridization using a mixture of radioactive labelled probesc-abl and bcr sequences were employed on a CML patient sample. Althougha translocation was said to be detected, Poisson analysis, a statisticalprocedure, was required to differentiate random from non-random silvergrain distribution after autoradiography. (Bartram et al., 1987).

Benn et al. (1987) relates the molecular genetic analysis of the bcrrearrangement in the diagnosis of CML. Analysis involved Southern blotsand radioactively labelled probes.

A single bcr-derived probe from which highly repetitive sequences wereremoved, was employed to detect the Ph¹ translocation in CML.Restriction fragment length polymorphisms (RFLP) were used to identifiedpatients affected with CML. Probes were used to map the chromosome 22breakpoints within the bcr region by Grossman et al. (1989). Twoseparate bcr-specific probes were used to detect rearrangements withinthe bcr region. Southern blots and RFLP were employed. (Hutchins et al.,1989).

Flow cytometry has been applied to detection and characterization ofdisease—linked chromosome aberrations (Gray et al., 1990). There is agreat need to improve methods of detecting specific chromosomeaberrations. Flow cytometry requires in vitro cell culture, expensiveequipment, and expertise in interpretation of statistical analyses ofresults. Therefore, it is not generally clinically useful.

Detection of aberrations by use of repeat sequence probes found nearcentromeres, generally alpha satellite probes, or whole chromosomeprobes not probes specific for genetic regions associated with diseases.Greater sensitivity and increased resolution is needed. Use of wholechromosome probes is generally limited to detection of aberrations thatoccur homogeneously in a cell population (Gray et al., 1990) and doesnot have the resolution to distinguish similar, but distinctbreakpoints. The present invention relates methods and compositions fordetection of chromosomal aberrations that need not be present in allcells of a sample. Compositions include novel probes that werespecifically designed to detect the BCR-ABL fusion gene in acute andchronic leukemias e.g. CML and ALL, and to determine molecular subtypes.

Methods using a plurality of probes to provide increased sensitivity andspecificity in detecting chromosomal aberrations, are also aspects ofthe present invention. These methods are particularly valuable in beingapplicable to interphase cells, thus avoiding the costly, laborious,time-consuming and often inconclusive cytogenetic analysis of metaphasechromosomes, and the expertise needed for flow cytometry. Not only arethe methods of the present invention easier to use, but these methods donot require invasive or risky techniques inflicted on patients, such asbone marrow sampling. However, the methods and compositions of thepresent invention may also be used on metaphase chromosomes or Southerblots.

SUMMARY

Substantial proportions of human diseases and malformations trace theiretiology, at least in part, to genetic factors. Some are inherited, someoccur during the development and life of the organism. Cancers, forexample, are associated with somatic mutations and/or chromosomalaberrations that may be specific for cancerous cells. Detection ofgenetic factors associated with particular diseases or malformationsprovides a means for diagnosis and treatment. For some conditions, earlydetection may allow prevention or amelioration of the devastatingcourses of diseases. For others, monitoring the course of the disease isuseful to determine treatment strategies. The methods and compositionsof the present invention provide multipronged reconnaissance into thegenetic material to determine if it harbors abnormal factors.

This invention concerns genetic factors in the form of chromosomalaberrations, that is, deviations from the number and structure ofchromosomes characterizing a species, and cell types within the species.In humans, for example, there are generally 46 chromosomes in somatic,i.e. non-germinal cells. These exist in 23 pairs, 22 of which are eachmatched by size and structural morphology. Structural morphology isrevealed by a variety of methods, for example, treatment of chromosomesto form distinguishable horizontal bands. Analysis of such patterns, andcomparison of the relative size of the chromosomes and positions of thecentromere, a constriction visible at metaphase of the mitotic cellcycle on each chromosome, allow identification and classification ofeach pair. Analysis of banding patterns also permits detection ofstructural aberrations both between and within chromosomes.

For purposes of the present invention, structural chromosomalaberrations which comprise a breakpoint fusion region with nucleic acidsequences flanking the breakpoint fusion, are of particular interest.Flanking regions should be within 800 kb or less so that there areinclude a particular breakpoint, yet are far enough on either side ofthe fusion so that they are not included in it. An aspect of the presentinvention is to detection aberrations which are not detectable byconventional metaphase cytogenetics using light microscopy.

Chromosomes contain linear sequences of DNA, a nucleic acid that is thegenetic determinative for most species (RNA is the genetic material insome lower organisms). Cloning technology has been developed which iscapable of isolating specific genes directly from the genome.

To identify specific genes, that is, specific nucleic acid sequences,specific probes may be used that react only with the particular sequenceof interest to seek it out from the vast excess of other sequences. Thereaction of probes and their matching (homologous) sequences, is termedhybridization—the joining of the probe and its match by hydrogen bonds.Laboratory methods related to cloning technology and other techniqueswell known to those of skill in the art, may be found in Maniatis (1982)and in Lewin (1987). Conditions of varying stringency are used dependingon the degree of homology required for a match. In examples disclosedherein, stringency conditions are set forth that are specific forhybridization to unique breakpoint provided in preferred embodiments.

The power of this approach in cytogenetic analysis comes from theincreasing availability of chromosome- or locus-specific-nucleic acidprobes. These fall into three general classes: 1) probes for sequencesthat are present in many copies on one chromosome, 2) composite probescomposed of many individual elements that are homologous to targetsequences distributed more-or-less continuously along an entirechromosome, and 3) probes homologous to a specific chromosome subregionor locus; for example, associated with a genetic disease. To use probesto detect chromosomal aberrations formed by breakage and reunion of,e.g., two chromosomes from different pairs (non-homologous), by probingthe sequence at the fusion of the breakpoints themselves, may provide aweak signal by which the hybridization is detected. This is because whena short sequence hybridizes, the signal may be too weak to be detected.If a probe is lengthened to provide greater signal intensity, it maybecome too large. This will be seen as a diffuse signal. Those of skillin the art will readily determine optimum probe size for a particularapplication using the guidelines disclosed herein.

A laboratory procedure that produces increased specificity and lowbackground, and one in which individual probes can be distinguished asseparate entities, is preferred. Thus, the labelling of probes is notamenable to current radioactive isotopic labels, but is more suitablyperformed with fluorescent and other non-isotopic, i.e. enzymatic orchemical labeling methods. For diagnosis using interphase cells, thatstage of cell division that most somatic cells sampled clinically arein, these labelling methods provide good enough intensity to bedetectable.

An upper limit on probe size for purposes of the present invention isbelieved to be about 200 kb of nucleic acids, that is, about 3 times thesize used in the examples disclosed herein. A goal in determiningsuitable sizes for probes is to detect doublets. Doublets are pairs ofdistinct probes in closer proximity than expected based on there normalchromosome locations in the absence of aberrations. To overcomelimitations inherent in some other techniques, this invention provides astrategy of multiple sorties into the genetic material using at leasttwo probes for separate, but related sequences; for example, one foreach of the flanking regions of a breakpoint at which fusion of twochromosomal segments has occurred. Moreover, this invention takesadvantage of probes large enough to give an intense signal yetspecifically targeted to a genomic sequence. To be distinguishable yetjuxtaposed at interphase, labelled flanking regions have to beapproximately within 800 kb.

The probes are preferably labelled so that their location in the geneticmaterial may be determined. The location is generally determined by useof a microscope. To avoid increased time and the usual problems andrisks associated with radioactive labels, fluorescent labels arepreferred. A separate color for each independent probe provides the mostinformation. e.g. red on one probe, green on the other.

The probes may be detected in situ, that is, without extraction of thegenetic material. In general, it will be the cell, or, morespecifically, the cell nucleus that will be viewed.

Although the cells may be analyzed in metaphase, a stage in celldivision wherein the chromosomes are individually distinguishable due tocontraction, the methods and compositions of the present invention areparticularly useful for interphase, a stage in cell division whereinchromosomes are so elongated that they are entwined as is a bowl ofspaghetti, and cannot be individually distinguished. At this stage thechromosomes may be referred to as chromatin.

An additional aspect of the invention is the use of genomic DNAfragments as probes, rather than fragments which correspond simply totranscribed/translated regions. By employing genomic fragments of up to100 kb, e.g., through the use of cosmid clones, it is possible to obtainmuch greater relative degree of hybridization with the chromosomal DNA,a particular advantage where a light-microscopic detection is envisionedsuch as in the preferred method in the present invention.

Using multiple probes, each with a distinguishable label, the overallpattern of the probes is used to assay for a breakpoint. Because allelicgenes exist on chromosome pairs, each labelled probe capable ofhybridizing to a sequence normally present on a specific chromosome,appear twice. If one member of each of two chromosomes is involved in atranslocation which moves the sequences hybridizing to the probestogether on one fusion chromosome, two of the different colored probeswill be in closer proximity to each other than expected if they maintaintheir original chromosome location, the other two will be more distant.

In particular aspects of this invention, specific disease entities areanalyzed. The hematological malignancies provide illustrativeembodiments as disclosed in the following sections. One of the mostclinically useful assays for chromosomal aberrations is cytogeneticanalysis directed at detection of the Philadelphia chromosome (Ph¹)which is associated with chronic myelogenous leukemia (CML), and otherhematological malignancies. The presence or absence of the Ph¹chromosome is a major diagnostic and prognostic aid. However, detectionby cytogenetic analysis and other available techniques is timeconsuming, laborious, and not completely accurate.

An aspect of the present invention concerns the use of DNA probes forthe direct detection of Philadelphia chromosomes in metaphase andinterphase cells using non-radioactive methods. The so-calledPhiladelphia chromosome is a chromosomal aberration which results from atranslocation between chromosome 9 and 22 which produces a longerchromosome 9 and shorter chromosome 22. The shortened chromosome 22,termed Ph¹, is generally diagnostic of certain types of leukemia,including in particular chronic myelogenous leukemia (CML), as well asvarious other leukemias. On a molecular level, it has been shown thatthe development of the Ph¹ chromosome includes a translocation of aportion of the c-abl oncogene into a breakpoint cluster region (bcr) ofchromosome 22, which can activate the ABL gene.

A variety of molecular methods are known for diagnosing this abnormalityin DNA or RNA extracted from cancer cells. The method of the presentinvention involves the use of a specific set of DNA probes, somecorresponding to the abl gene, and some corresponding to the bcr gene.This specific set of probes is hybridized in situ to fixed cells of asample from an individual suspected of being affected. The ABL and BCRspecific probes are preferably labeled with separate fluorescein tags(e.g., biotin plus fluorescein-labeled avidin, or digoxigenin-labeledprobes). Therefore, upon hybridization, both sets of labeled probes willhybridize to an a translocated chromosome 9 producing a two colordoublet, whereas only the ABL specific probe will hybridize tochromosome 9 in non-affected individuals. The use of a visuallydetectable label allows a means of assessing the presence of the Ph¹chromosome through the application of light microscopy, providing asignificant advantage in terms of expertise required to carry out theassay. The methods are simpler and more rapid than previously available.

Probes developed as an aspect of the present invention include threeprobes that are particularly useful for detection of hematopoieticmalignancies, notably chronic myelogenous leukemia (CML) and acutelymphoblastic leukemia (ALL). The novel probes are designated: PEM12,c-H-abl and MSB-1. These probes are specific for regions of the BCR gene(MSB-1 and PEM12) and a region at the ABL gene (c-H-abl). The BCR andABL regions are those which flank the breakpoint fusion region in thePh¹ chromosome associated with leukemias.

FISH was used to detect the Ph¹ chromosome or its genetic equivalent asthe fusion of BCR and ABL probes labeled with two colors. The method wassuccessfully used in interphase cells of ALL patients. This method,using only two probes, only detects the p210 subtype of BCR-ABL genefusions, whereas the majority of Ph¹-chromosome-positive ALL casescontain the p190 fusion. For this reason, a combination of three probesused in pairs was developed that could detect both the p190 and p210molecular subtypes. Methods using these combined probes are useful forPh¹ chromosome detection by FISH in ALL. Capabilities and limitations ofprobes and combinations of probes in the clinical setting were assessedand shown to provide improvements over previous assays for leukemias, inparticular cytogenetic analysis of metaphase chromosomes. Although theembodiments herein relate to detection of chromosomal aberrations inleukemias, the probes may be specifically tailored to meet clinicalneeds for the diagnosis of any chromosomal aberration, as they have fortranslocations in leukemias. It is only necessary to be able todetermine breakpoint regions and to develop probes to those regions.

The Philadelphia (Ph¹) chromosome is also an important prognosticindicator in acute lymphocytic leukemia (ALL). Present in 30% of adultand 5% of pediatric cases, its presence portends a short remissionduration and poor survival, despite improvements in therapy as in CML.It is a derivative of a translocation between chromosomes 9 and 22, andresults in the fusion of a part of the ABL proto-oncogene on 9q withpart of the BCR gene on chromosome 22. Molecular analysis shows it ismuch more heterogeneous because the BCR breakpoints are variable.Cytogenetic diagnosis of the Ph¹ chromosome in ALL is possible in only70% of cases because of the failure to obtain adequate metaphases. Thenew technique of fluorescence in situ hybridization (FISH) offers theadvantage of allowing the diagnoses of chromosomal abnormalities ininterphase cells, thus overcoming the problem of metaphase preparations.

Using dual-color FISH with probe combinations specifically tailored toflank the breakpoints in the two types of the BCR-ABL fusion genes p210and p190, the presence or absence of the Ph¹ chromosome in interphasecells was determined from 5 ALL patients, two ALL-derived cell lines,and normal lymphocytes and specified its molecular subtype when present.The method proved accurate for detection in all cases and for subtypingin 7 of 8 of the cases examined. The sensitivity and specificity forassessing the Ph¹ status of individual cells were low, but results wereunequivocal when several cells were examined in a sample.

As can be seen from the following descriptions and examples, the methodsdisclosed may be performed by a pathologist on routine examination ofblood and tissue samples.

FIGURES

FIG. 1: is a schematic representation of a reciprocal translocationbetween chromosomes No. 9 and 22.

FIG. 2: is a schematic representation of the location of probes used fordual-color FISH on the normal BCR gene and on BCR-ABL fusion genesubtypes.

FIG. 3: illustrates expected signal (labelling) patterns associated withdifferent molecular subtypes of the Ph¹ in interphase cells usingtwo-color FISH with two different probe combinations used.

FIG. 4: is a restriction enzyme map of part of the human BCR gene fromchromosomal 22.

FIG. 5: is a restriction enzyme map of the human c-abl region fromchromosome 9.

DETAILED DESCRIPTION OF THE INVENTION

The following examples, materials and methods provide embodiments of theinvention.

EXAMPLE 1 Detection of Ph¹ IN CML

Two-color FISH with the abl (red) or bcr (green) probe to normal G₁interphase nuclei in most cases resulted in two red and two greenhybridization signals that were well separated and randomly distributedaround the nucleus. In a few cells, two doublet hybridization signalswere detected, probably as a result of hybridization to both sisterchromatids of both homologs in cells that had replicated this region ofDNA (those in the S or G₁ phase of the cell cycle).

Depending on the exact positioning of the breakpoints in the leukemicclone, the genetic rearrangement of CML brings the binding sites of thebcr and abl probes to within 25 to 225 kb of each other on an abnormalchromosome. Dual-color hybridization with abl and bcr probes tointerphase CML cells resulted in one red and one green hybridizationsignal located randomly in the nucleus, and one red-green doublet signalin which the separation between the two colors was <1 μm. In some cases,the red-green doublet appeared yellow. The randomly located red andgreen signals are likely due to hybridization to the abl and bcr geneson the normal chromosomes, and the red-green doublet signal tohybridization to the bcr-abl fusion gene. The distance between the redand green components of the fusion signal is consistent with interphasemapping studies. Those studies have shown that DNA sequences separatedby less than 250 kb should be within 1 μm of each other intwo-dimensional interphase nuclei. Since the positions of the bcr andabl hybridization sites are distributed apparently randomly over thetwo-dimensional nucleus images in normal cells, it is not surprisingthat some normal cells will have red and green signals separated by <1μm. Such false positive cells were found at a frequency of about 1% (9of 750 cells pooled from four normal individuals). The highest frequencyof false positive fusion signals for an individual case was 3 of 150cells analyzed. Thus, with the use of this probe placement strategy,these results set a practical limit of about 1% for the detectablefrequency of CML cells in a population.

Hybridization results for seven samples from six CML cases and data fromPCR, Southern, and chromosome banding analysis are presented in Table 1.In all six cases red-green hybridization signals separated by <1 μm inmore than 50% of nuclei were present. This was the case in three casesfound to be Ph¹-negative by banding analysis (CML-4, CML-5, and CML-6).In most cases, the fusion event was visible in virtually every cell. Onecase (CML-6) showed fusion signals in almost every cell despite the factthat PCR analysis failed to detect the presence of a fusion mRNA andbanding analysis did not reveal a Ph¹. Hybridization to metaphase cellswas performed in three cases (CML-1, CML-4, and CML-5). Red and greenhybridization signals in close proximity on a single small acrocentricchromosome were present in all three. In two cases (CML-1 and CML-4)scored as t(9:22)(q34:q11) by banding analysis, the red-green pair wasin close proximity to the telomere of the long arm of a smallacrocentric chromosome as expected for the Ph¹.

From the results of banding analysis one case (CML-5) was suspected tohave an insertion of chromosomal material at 22q11. Two-colorhybridization to metaphase cells from this case showed the red-greenpair to be centrally located in a small chromosome. This result isconsistent with formation of the bcr-abl fusion gene by an interstitialinsertion. Fusion genes are not always detectable by cytogenetic bandinganalysis of metaphase chromosomes.

In one case (CML-1), two pairs of red-green doublet signals were seen in3 of 150 (2%) interphase nuclei. This may indicate a double Ph¹ (ordouble fusion gene) in those cells that was not detected by bandinganalysis, which was limited to 25 metaphase cells. The acquisition of anadditional Ph¹ is the most frequent cytogenetic event accompanying blasttransformation, and its cytogenetic detection may herald diseaseacceleration.

Samples CML-3a and CML-3b represent an analysis of peripheral blood andbone marrow, respectively, from the same patient. The percentage ofbcr-abl fusion-positive cells was higher in the bone marrow thanperipheral blood.

TABLE 1 A summary of cytogenetic, FISH, and other analyses of bcr-ablrearrangements in six CML cases. CML-1 and CML-5 were bone marrowsamples from patients with chronic phase CML who were receiving notreatment. CML-3a and CML-3b were from peripheral blood and bone marrow,respectively, of a CML patient in blast crisis, who was receivinghydroxyurea, CML-2 was from bone marrow in a blast crisis CML patient.CML-4 was bone marrow from a CML patient in blast crisis and receivingno treatment. CML-6 was from bone marrow in a chronic phase CML patientreceiving hydroxyurea. Hybridization to metaphase cells was done oncases CML-1, CML-4, and CML-5. CML-1 and CML-4 both showed fusion genesignals localized near the end of a small acrocentric chromosomeconsistent with a classic Ph¹ resulting from a reciprocal translocation.CML-5 showed an interstitial fusion signal on 22q consistent with thebcr-abl fusion gene resulting from an insertional event. F, fusion; N,normal; D, double fusion; NI, not interpretable; and ND, not done.Interphase Patient Cytogenetics FISH Other Analysis CML-1 46, XX, t(9;22)(q34; q11)  80% F ND  2% D  18% NI CML-2 46, XY, t(9; 22)(q34; q11) 60% F PCR^(b)  40% NI CML-3a 46, XY, t(9; 22)(q34; q11)  75% F PCR^(a) 25% N CML-3b 46, XY, t(9; 22)(q34; q11) 100% F PCR^(a) CML-4 47, XY,+8, del(22)(q11) 100% F PCR^(b) CML-5 46, XY, ins(22; 9)(q11; q34q34)100% F PCR^(b) CML-6 46, XY, t(5; 9)(q13; q34) 100% F PCR^(b)Southern^(c) ^(a)See Materials and Methods, PCR, Kohler. ^(b)SeeMaterials and Methods, PCR, Hogewisch ^(c)See Materials and Methods,Southern Blot.

EXAMPLE 2 Detection of Ph¹ in all

Location of probes used for dual-color FISH on the normal BCR gene andon BCR-ABL fusion gene subtypes is shown in FIG. 2. The normal BCR gene,and the two subtypes of BCR-ABL fusion gene are schematicallyrepresented. Black vertical bars represent BCR exons, with the firstexon indicated by Roman numeral I, and the breakpoint cluster regionindicated by “bcr.” Open vertical bars represent ABL exons, and thesecond exon is indicated by Roman numeral II. The diagrams are not toscale; the approximate total distance of the BCR gene is 130 kb; thetotal extent of the ABL regions depicted here is 40 kb.

A. The normal BCR gene showing the approximate location of the MSB-1probe, and the PEM12 probes (not to scale).

B. The p210 subtype of the BCR-ABL fusion gene as found on the Ph¹chromosome, showing the approximate location of the MSB-1 probe and thePEM12 probe, relative to the c-H-ABL probe. The jagged line indicates apossible translocation breakpoint.

C. The p190 subtype of BCR-ABL fusion gene, with a representativebreakpoint indicated by the jagged line and the approximate locations ofthe MSB-1 and c-H-abl probes in this gene. The breakpoint is locatedproximal to the PEM12 probe, and it therefore translocates to the 9q+chromosome and is separated from the ABL sequences.

Detection of the Ph¹ in interphase cells using two-color FISH with probecombinations used and expected signal patterns given by differentmolecular subtypes is shown in FIG. 3. Open circles represent the greensignal of fluorescein-conjugated anti-digoxigenin antibodies to detectthe MSB-1 or PEM12 probes and hatched circles represent the red signalof avidin — Texas red used to detect the biotinylated probe c-H-abl.

A. The p190 fusion gene will show a pattern of doublets with theMSB-1+c-H-abl combination but result in separated doublets withPEM12+c-H-abl.

B. The p210 fusion gene show doublets with both probe combinations.

C. Normal or Ph¹-negative ALL cells show separated signals with bothprobe combinations.

FIG. 4: illustrates a restriction enzyme map of part of the human BCRgene from chromosomal 22. B=BAM H1; Bg=Bg111; E =EcoR 1; H=Hin dill. Thedark area in brackets below the map indicates the PEM12 region, anapproximately 18 kb insert in lambda phage. Arrows indicate breakpointsin ALL and CML.

FIG. 5: illustrates a restriction enzyme map of the human c-abl regionfrom chromosome 9. The arrow below the map indicates the c-hu-abl cosmidregion, approximately a 30 kb insert.

Detection of Fusion Gene Subtypes

The detection of the two BCR-ABL fusion gene subtypes is outlined inFIG. 3. The molecular basis of the Ph¹ chromosome is a translocationbetween the long arms of chromosomes 9 and 22, t(9;22)(q11;q34) (Rowley,1973) (FIG. 1 where the hatched chromosome material was originally onchromosome 9, the clear on chromosome 22; after breakage and reunion,two derivative chromosomes are formed, the small being the Ph¹chromosome which juxtaposes part of the ABL protooncogene on chromosome9q34 (Kurzrock et al., 1988) next to part of the BCR gene on chromosome22q11.) The resulting fusion gene is transcribed and translated toproduce a chimeric protein. Two types of BCR-ABL fusion genes exist inALL. One type has a BCR breakpoint in the limited region of the M-bcr(Groffen et al., 1984) and produces a 210 kd protein, designated p210.This is the type of fusion gene found in virtually all cases of CML. Theother type of fusion gene has a BCR breakpoint in the large region ofthe BCR first intron, (Rubin et al., 1988; Heisterkamp et al., 1988) andit produces a 190 kd protein, p190. This type of fusion gene accountsfor 75% of the Ph¹ positive cases of ALL, the remainder having the p210rearrangement.

A map of the normal BCR gene (Hooberman et al., 1989) and the two typesof fusion genes, with probe localizations, is shown in FIG. 2. Thec-H-abl probe contains the last exon of the ABL gene, which is anecessary part of the BCR-ABL fusion gene. The MSB-1 probe contains thefirst exon of the BCR gene, while PEM12 lies immediately 5′ of theM-bcr. Both the PEM12+c-H-abl and MSB-1+c-H-abl probe combinationsproduce doublets when a p210 type of fusion gene is present, becauseboth of these regions of the BCR gene are retained on the Ph¹chromosome. When a p190 fusion gene is present, however, the breakpointexists between these two probes, so that only MSB-1 is retained on thePh¹ chromosome to fuse with c-H-abl, while PEM12 remains on the 9q+chromosome. Consequently, doublets are seen with MSB-1+c-H-abl but notwith PEM12+c-H-abl.

In FIG. 2A, the regions of the BCR gene to which the two probes, MSB-1and PEM12 hybridize, are shown. A labelled MSB-1 probe contacted tochromosomes which hybridized to a normal human chromosome No. 9, wouldbe detected in association with that chromosome. Similarly, a labelledPEM12 probe would hybridize with a chromosome No. 9 and show associationwith it by detection of the label.

The nucleic acid sequence of the ABL gene is normally on chromosome No.22. The probe c-H-abl will hybridize to a region of the ABL gene asshown in FIG. 2B. Also shown in FIG. 2B are the locations of the p210subtype of the fusion gene formed after breakage and reunion ofchromosomes No. 9 and 22 to form a fusion gene. This fusion gene isdesignated p210 because it is capable of being expressed as a fusionprotein with a molecular weight of approximately 210 kd as determined bySDS gel electrophoresis.

Application of both probe combinations thus permits the both thedetection of the BCR-ABL fusion gene, and specification of the subtype.A schematic diagram of expected results in interphase is shown in FIG.3. Because the ABL breakpoint is variable, the probe separations may befrom 25 to over 200 kb. In metaphase in situ hybridization, this rangeof distances will cause the probe signal to fuse, but in interphase insitu hybridization the two colors can be resolved.

If combinations of two dual labelled probes are added to a preparationcontaining the p210 subtype of fusion gene, a pattern in interphase cellnuclei will appear as showing a dual labelled doublet where the fusiongene is located, and two single labels where the normal (untranslocated)chromosome Nos. 9 and 22 are located (FIG. 3B). In FIG. 3B, theappearance of doublets of MSB-1+c-H-abl, and PEM12+c-H-abl, are shownschematically.

The presence of the p190 subtype of fusion gene can be distinguishedfrom the presence of the p210 fusion gene, or the absence of fusiongenes of this type in normal cells (FIG. 3), because the p190 fusiongene does not have a PEM12 site (FIG. 2C). Therefore, as shown in FIG.3A, labelled doublets reflecting probe associations are only expectedwith the combination of probes MSB-1+c-H-abl. Contacting cells with theprobe combination PEM12+c-H-abl will not yield doublets, and shouldproduce an appearance at interphase of a cell pattern undistinguishablefrom normal cells. (Compare FIGS. 2A and 2C).

Five cases of newly-diagnosed adult ALL, were analyzed using the methodsof the present invention. Results are shown in Table 2. Cases 1 to 3were peripheral blood samples, but all had greater than 50% blasts inthe sample; cases 4 and 5 were bone marrow samples. Determination of thepresence or absence of a fusion gene agreed with the molecular resultsin each case. The two negative cases were easily scored. Among theBCR-ABL positive cases, the subtype was obvious in patients 3 and 5, butindeterminate in patient 2. For patient 2, the MSB-1+c-H-abldetermination was unequivocal, and many cells even contained twodoublets; however, the result for the PEM12+c-H-abl combination wasindeterminate, and no cells were observed which contained multipledoublets. Several subsequent attempts to repeat the assay with thePEM12+c-H-abl combination failed to resolve the issue.

Variability between runs was apparent. The best hybridization resultsallowed the interpretation of one cell in three. Any hybridization inwhich no more than one cell in 8-10 showed results was discarded.Factors which affected the quality of hybridization results includedquality of the sample, with best results from freshly fixed (<1 day infixative) samples showing even, rather than clumped, chromatincondensation, and minimal residual cytoplasm. Other factors included thehybridization efficiency of the probes, and the ability to visualize thedoublets, which were sometimes difficult to resolve into two differentcolors. The final problem was greatly improved by a change fromfluorescein-tagged anti-digoxegenin Fab fragments to new, polyclonal,whole-antibody anti-digoxigenin antibodies (both by BoehringerMannheim). Of the three probes, the c-H-abl cosmid produced the mostevaluable cells, followed by PEM12, with MSB-1 as the least efficient ofthe probes. Of hybridizations which were discarded for failure of oneprobe to hybridize, the most often to fail was MSB-1.

The presence or absence of the BCR-ABL fusion gene were correctlyassigned in 2 cell lines and 5 clinical specimens of ALL. The molecularsubtype was easily specified for most of these cases except for case 2,in which the relatively high false positive rate (17%) madeinterpretation difficult.

The analysis was performed directly on interphase specimens ofperipheral blood or bone marrow, obviating the need for cell culture andmetaphase preparation. The ability to detect chromosomal abnormalitiesin interphase cells has an important impact on ALL, because failure toobtain adequate metaphases is one of the most common reasons forcytogenetic failure in this disease. Another important feature of FISHcompared to conventional cytogenetics is that it directly detected theimportant molecular events—the BCR-ABL gene fusion subtype—rather thanmerely the presence of chromosomal aberration. In this regard, itcompares favorably to molecular methods of diagnosis. The BCR-ABL fusiongene is an important clinical finding in ALL, and FISH is a viableoption for its detection. The limitations and capabilities of thedisclosed methods were evaluated in a clinical setting.

Although the ability to diagnose a case where more than one cell wasavailable for analysis, which will generally be the situation presented,was excellent, the ability to detect the Ph¹ in an individual cell wasrather more limited. The rate of detection of a positive signal waslower than might be predicted. In the cell lines, which are believed tocontain 100% Ph¹ positive cells, the range of detection was 46-83%positive cells, with an overall average of 68%. In the patient samples,the average number of cells scored as positive was lower; however, theseare heterogeneous populations of cells, and lower FISH results forpatients relates to the lower percentage of malignant cells in thesamples. There will generally be both normal and malignant cells in asample, and the percentages will vary from patient to patient, and evenwithin samples from the same patient.

Similarly, the rate of detection of a false positive doublet signal is 3to 10% of cells of normal donors.

In summary, interphase detection of FISH with these probe combinationsare an accurate method of detecting the presence of the Ph¹ chromosomein ALL, and may be a technique which will afford this diagnosis innearly 100% of ALL patients.

EXAMPLE 3 Detection of Ph¹ in a Cell Line

Cell Lines and Normal Lymphocytes

The two probe combinations were tested on preparations of normallymphocytes, a cell line with a known p210 gene fusion (BV173), and acell line with a known p190 gene fusion (SUPB13). The results of thesedeterminations, performed blindly, are shown in Table 3. It was foundthat the presence or absence of either subtype of the BCR-ABL fusiongene could accurately be assessed. However, none of the cell lines gave100% fusion gene-positive cells, as would be expected from a homogeneouspopulation of cells. The percent of cells scored as positive ranged from46 to 83% of cells with recognizable signals, indicating a falsenegative rate of 17 to 56%. The false negatives were highly dependent ondegree of background staining, sample quality, hybridization efficiencyof the probes, and experience. The rate lowered as the observer becamemore experienced.

The false positive rate, the number of cells with doublets in normallymphocytes, or of SUPB13 cells containing PEM12+c-H-abl doublets,ranged from 3 to 10%. Based on this experience, it was found useful todefine the positive and negative patient cases relative to normalcontrols run at the same time. A case was defined as positive when thedoublets were present at two-and-a-half times the rate found withnegative controls, and as negative if the rate of doublets was similar(within 5%) to the normal lymphocyte results. Any findings in betweenwere scored as indeterminate.

Multiple red-green hybridization sites along both arms of a singleacrocentric chromosome were detected in simultaneous hybridization ionswith abl and bcr probes to metaphase cells of the CML-derived cell lineK-562 . Hybridization to interphase nuclei demonstrated that the red andgreen signals were localized to the same region of the nucleus. This isconsistent with their being present on a single chromosome. Eight to 16hybridization pairs were seen in each of 250 nuclei enumerated,indicating corresponding amplification of the bcr-abl fusion gene.Fusion gene amplification was not seen in any of the normal controls orCML patients analyzed. These findings are consistent with previousSouthern blot data showing amplification of the fusion gene in this cellline.

In summary, dual-color FISH analysis of interphase cells from seven CMLand four normal cell samples with abl and bcr probes suggests theutility of this approach for routine diagnosis and clinical monitoringof CML. A significant advantage of this technique is the ability toobtain genetic information from individual interphase or metaphase cellsin less than 24 hours. Its application is not limited to cells that,fortuitously or through culture, happen to be in metaphase; it can beapplied to all cells of a population. The genotypic analysis can beassociated with cell phenotype, as judged by morphology or othermarkers, and this makes possible the study of lineage specificity ofcells carrying the CML genotype, as well as assessment of the frequencyof cells carrying the abnormality. Moreover, counting of hybridizationspots allows the determination of the degree of bcr-abl geneamplification in the K-562 cell line. It is possible that this analysismay be further developed using quantitative measurement of fluorescenceintensity.

TABLE 2 EVALUATION OF THE Ph¹ CHROMOSOME IN LEUKEMIA SPECIMENS USINGDUAL-COLOR FISH. FISH Determinations* Probe (combinations) CombinedPatient MSB1 + PEM12 + FISH Molecular Case Data c-H-abl c-H-abl ResultsFindings 1. 16F 92/100 (8%) 14/107 (13%) Ph¹ negative BCR-ABL negative2. 63F 36/114 (31%)** 19/110 (17%) Ph¹ positive BCR-ABL positiveindeterminate p190 subtype 3. 61M 54/99 (55%) 83/140 (59%)** Ph¹positive BCR/ABL positive p2210 subtype p210 subtype 4. 39M 2/59 (3%)11/118 (9%) Ph¹ negative BCR-ABL negative 5. 38M 46/104 (44%) 9/108 (8%)Ph¹ positive BCR-ABL positive p190 subtype p190 subtype *Results arepresented as number of cells with doublets/total number of evaluablecells (% cells with doublets) **Multiple doublets were observed in somecells in these cases.

TABLE 3 EVALUATION OF PROBE COMBINATIONS ON Ph¹ CHROMOSOME-POSITIVE CELLLINES AND NORMAL LYMPHOCYTES. SAMPLE MSB-1 + c-H-abl (%)* PEM12 +c-H-abl (%)* SUPB13 1. 56/104 (46%) 1.  7/119 (6%) 2. 48/62 (77%) 2. 7/101 (7%) 3. 71/22 (58%) BV173 1. 80/96 (83%) 1. 43/73 (59%)  2. 76/97(78%) 2. 80/102 (78%)  Normal 1.  3/120  (3%) 1.  8/106 (8%) Lymphocytes2. 12/121 (10%) 2.  8/109 (7%) *Results expressed as number of cellswith a doublet/total number of evaluable cells (percent positive cells).Each line represents a separate run.

Materials and Methods

Patient Samples

Cases were selected from newly diagnosed ALL samples referred to theinventor's laboratory for pulsed field gel electrophoresis (PFGE) andSouthern molecular analysis for the BCR-ABL fusion gene. Peripheralblood was anti-coagulated with EDTA (lavender-top tubes) and bone marrowwith heparin (green-top tubes). Buffy coats were removed from thesamples and incubated with NH₄Cl (0.135M in 0.005M Tris HCl ph 7.6) tolyse red cells. After several washes in Hank's balanced salt solution(HBSS, Gibco), the cells were counted and viability assessed. Cells forFISH were allowed to “rest” in the final wash solution of HBSS for a fewhours at 4° C. Cells for molecular analysis were embedded in agaroseplugs according to previously described procedures. (Hooberman et al.,1989). In two cases (Patients 2 and 5), cells for FISH were thawed fromliquid nitrogen storage, and incubated without stimulation or colcemidarrest for 3-24 hours prior to harvest.

Two lymphoblastoid cell lines, SUPB13 (Rubin et al., 1988), positive forthe p190 type of fusion gene, and BV173 (Westbrook et al., 1988),positive for p210, were used. They were grown in RPMI 1640 (Gibco) with10% fetal bovine serum and penicillin-streptomycin (Sigma, St. Louis,Mo.). Peripheral blood lymphocytes, obtained from normal healthy donorswere stimulated with phytohemagglutinin (PHA, 1 mg/ml,Burroughs-Welcome) and cultured for 72 hrs. Cell lines and stimulatedlymphocytes were incubated at 37° C. in 5% Co₂. Twenty-five and 5minutes prior to harvest, Colcemid (Gibco) was added to the cultureflasks to a final concentration of 0.1 ug/ml to produce metaphasearrest.

Probes

1. Description

PEM12 is a phage clone containing an 18 kb human genomic insert inEMBL3. It contains part of a sequence of the major breakpoint clusterregion (M-bcr) of chromosome 22 and extends 5′ of it. M-bcr is an areawherein breakpoints cluster within the BCR gene.

The c-H-abl probe is a cosmid approximately 40 kb in size with a 35 kbhuman insert in pCV105, and 5 kb of vector, specific for the 3′ end ofthe ABL gene. The cosmid was isolated from a cosmid library 105SL/108Kprovided by Dr. Chris Y-F Lau. This library is generally available forresearch use and has been described in Proc. Natl. Acad. Sci. USA80:5225 (1983). Cosmids were hybridized with probes containingcontiguous fragments of the genes of interest, here the ABL genesequences. The procedure was to use probes of increasing size.

MSB-1 is a phage clone with an 18 kb fragment of human DNA from thefirst exon of the BCR gene cloned into EMBL3.

The PEM12 and MSB-1 probes were labeled with digoxigenin-11-dUTP(Boehringer Mannheim) and the c-H-abl cosmid was labeled withbiotin-11-dUTP (ENZO) by nick translation, using reagents supplied byENZO Diagnostics. The probes were combined with each other in twocombinations: MSB-1+c-H-abl and PEM12+c-H-abl. Fifty ng of c-H-abl wascombined with 150-170 ng of PEM12 or MSB-1 and 1 μg of human placentalDNA. The mixture was brought to a total of 10 μg of DNA/per slide withsalmon sperm DNA. The probe combinations were then ethanol precipitatedand redissolved in a hybridization solution of 50% formamide/10% dextransulfate in 2×SSC, heated to 70° C. for 5 min., then incubated at 37° C.for 15-30 min. prior to application to slides.

2. Preparation

The Sequence of the BCR and ABL genes are available in the GeneBank.™The sequences selected for use as probes may be amplified, e.g., by PCRwhich is well known to those of skill in the art, and used to screenlibraries. (Maniatis, 1982).

Ranges of preferred probe sizes and distances from the breakpoint-fusionarea, are disclosed in previous sections.

Slide Preparation and Hybridization

Cell lines, normal lymphocytes, or patient cells were pelleted bycentrifugation (1000 rpm for 10 min.), and treated with hypotonic KC1(0.075M), for 12 min. at 37° C. They were resuspended in 3:1methanol:acetic acid for fixation, and stored at 4° C. until slides wereprepared, usually one to ten days. The samples were pelleted again andwashed three times in fresh 3:1 methanol:acetic acid fixativeimmediately prior to dropping onto slides pre-cleaned with 95% alcohol.Slides of the samples were stored desiccated at 4° C. until use.

The slides were baked on a slide warmer for 4 hrs. at 65° C. They wereincubated in an RNase solution, 100 μg/ml in 2×SSC for 1 hr. at 37° C.,then washed 4 times in 2×SSC, 2 min. each. Next, they were passedthrough a graded alcohol series (70%, 80%, 95%), 2 min. each and allowedto air dry.

Denaturation of cellular DNA was performed in 70% formamide in 4×SSC for2 min. at 70° C., and the graded alcohol series and air drying wererepeated. A gentle proteinase K digestion, 60 ng/ml in 20 mM Tris/2 mMCaCl₂ at 37° C. for 8 min. (Pinkel et al., 1986) was followed by a thirdgraded alcohol series and air drying. The slides were warmed to 37° C.and held there until the probe hybridization mixture was applied. Ten μlof the probe mixture was applied to each slide, the area ofhybridization was covered with 22×22 mm coverslip, sealed with rubbercement and placed on a hotplate at 90° C. for 2 min. Two slides fromeach case were hybridized, one for the MSB-1/c-H-abl, and one for thePEM12/c-H-abl combination.

Detection of Hybridization

Detection steps are essentially those described by Trask et al. (Trasket al., 1991) with minor modifications. Following overnight incubationat 37° C. in a moist chamber, the coverslips were removed and the slideswashed 3 times in 50% formamide/4×SSC, 5 min. each at 40° C. To blocknonspecific binding, the slides were incubated for 5 min. at roomtemperature with 100 μl of 3% bovine serum albumin (BSA) (Sigma, St.Louis, Mo.) in 4×SSC under a coverslip.

The first detection reagent, avidin-Texas red (Vector) diluted in 3%BSA/4×SSC (2.5 ug of fluorochrome per ml of diluent) was applied, thecoverslip was replaced, and the slides were incubated at 37° C. for 1hr. The slides were washed 3 times, in 4×SSC, 4×SSC/0.1% Triton X, andPN (0.1M NaH₂PO₄/NaH₂PO₄ buffer pH 8/0.1% NP-40), sequentially, for 5min. each.

A second blocking step was performed with PMN (PN+5% non-fat drymilk+0.05% sodium azide, centrifuged to remove milk solids), 100 μl wasplaced under a coverslip for 5 min. at room temperature. The seconddetection and amplification reagent, anti-digoxigenin polyclonalantibody (Boehringer Mannheim) and biotinylated anti-avidin (Vector),was applied in a 1:25 dilution in PMN (100 ul/slide) and the slides wereincubated again at 37° C. for 1 hr. Three washes, 5 min. each, in PNfollowed. The PMN block step was repeated, and the third fluorescentreagent, avidin-Texas red and fluorescein-conjugated rabbit anti-sheepantibody (Vector) a 1:50 dilution, in PMN, was incubated at 37° C for 1hr. The final washes were PN×2, then 4×SSC/0.1% Triton X once for 5 min.A brief (1-2 min.) bath in DAPI (diamidino-2-phenyl-indole,dihydrochloride; (Sigma, St. Louis, Mo.)) 200 ng/ml in 4×SSC/0.1%TritonX, was followed by a rinse in 4×SSC. The slides were thencoverslipped with a DABCO antifade solution (diazabicyclooctane, Sigma)(90% glycerol/ 2.3% DABCO in 20 mM

Tris pH 8.0), and stored desiccated in light-tight boxes at 20° C. untilreviewed (usually less than three days later).

Molecular Analysis

Molecular analysis for the presence or absence of the BCR-ABL fusiongene and its subtype was performed by a combination of pulsed field geland Southern blot, as described previously (Hooberman et al., 1989). Allcases were reviewed by one observer without knowledge of the FISHresults.

Interpretation of Slides for Probe Hybridization

The slides were viewed with a Zeiss standard 16 microscope equipped forepifluorescent illumination and a set of dual band-pass filters (OmegaOptical). All samples were coded so that the observer did not know theresults of the molecular studies at the time of review. At least 100interphase cells were scored for the presence or absence of a red-greendoublet. A doublet was defined as red and green signals lying with adistance of 1 diameter of a signal, approximately 1 micron. Brightyellow signals which could not be resolved into red and green were notcounted as doublets. A slide was discarded if failure of hybridizationof one or both probes was apparent. When both probe combinations for acase had been scored, an assignment of the presence and subtype offusion gene was made.

Hybridization of Nucleic Acid Sequences

CML-3b and CML-6: Five to ten drops of marrow diluted with phosphatebuffered saline (PBS) to prevent clotting were fixed in methanol/aceticacid and dropped on slides.

CML-1, CML-2, CML-4, and CML-5: Peripheral blood or bone marrow, orboth, was cultured in RMPI 1640 supplemented with 10% feral bovineserum, an antibiotic mixture (gentamicin 500 μg/ml), and 1% L-glutaminefor 24 hours. Cultures were synchronized according to J. J. Yunio and M.E. Chandler, Prog. Clin. Path. 7, 267 (1977), and chromosomepreparations followed L. M. Gibas and Jackson, Karyogrom 9 (1986).

CML-3a: Peripheral blood was centrifuged for 5 min. at 1100 rpm, thebuffy coat was pipetted off and diluted with the same volume of PBS,spun down, fixed in methanol/acetic acid, and dropped on slides.Hybridization followed procedures described by Pinkel et al., Proc.Natl. Acad. Sci. U.S.A. 45 9138 (1983), Trask et al., Genomics 6:710(1989) and Lawrence, Villaive, and Singer, Cell 42, 51 (1988), withmodifications.

The bcr probe was nick-translated (Bethesda Research LaboratoriesNick-Translation System) with digoxigenin 11-dUTP (deoxyuridine5′-triphosphate) (Boehringer Mannheim Biochemicals) with an averageincorporation of 26%. The abl probe was similarly nick-translated withbiotin-11-dUTP (Enzo Diagnostics). Cells were thermally denatured at 72°C. for 5 min., dehydrated in an ethanol series, air-dried, and placed at37° C. A hybridization mixture (10 μl) containing each probe (2 ng/ul),50% formamide/2×standard saline citrate (SSC) 10% dextran sulfate, andhuman genomic DNA (1 mg/ml, sonicated to 200 to 600 bp) was heated to70° C. for 5 min. incubated for 30 min. at 37° C., placed on the warmedslides, covered with a 20 mm by 20 mm cover slip, sealed with rubbercement, and incubated overnight at 37° C. Slides were washed three timesin 50% formamide 2×SSC for 20 min each at 42° C., twice in 2×SSC at 42°C. for 30 min. each, and rinsed at room temperature in 4×SSC. Allsubsequent steps were performed at room temperature.

Slides were blocked in 100 μl of 4×SSC/1% bovine serum albumin (BSA) for5 min. under a coverslip. The biotinylated abl probe was detected byapplying 100 μl of Texas red-avidin (Vector Laboratories, Inc.), 2 μg/mlin 4×SSC/1% BSA) for 45 min. The slides were washed twice for 5 min. in4×SSC/1% Triton X-100 (Sigma). The signal was amplified by applyingbiotinylated goat antibody so avidin (Vector Laboratories, Inc., 5 μg/mlin PNM o.1M M NaH₂PO₄/0.1 M Na₂HPO₄, pH 8 (PN) containing 5% nonfat drymilk and 0.02% sodium amide and centrifuged to remove solids), washedtwice in PN for 5 min., followed by another layer of Texas red-avidin inPNM. The digoxigenin-labeled bcr probe was detected by incubation withsheep antibody to digoxigenin (obtained from D. Pepper, BoehringerMannheim Biochemicals, Indianapolis, Ind.; 18.4 μg/ml in PNM) for 30min, washed twice in PN for 5 min., followed by a rabbit-antibody tosheep conjugated with FITC (Organon Teknika-Cappel, 1:50 in PNM). Afterwashing twice for 5 min. in PN, the signal wa amplified by applying asheep antibody to rabbit immunoglobulin G(IgG) conjugated to FITC(Organon Tekniks-Cappel, 1:50 in PNM). The slides were then rinsed inPN. Slides were mounted in 10 μl of fluorescence body solution Johnsonand Nogueria, J. Immunol. Methods 43, 349 (1981) containing:4′,6-amidino-2-phenylindols (DAPI) at 1 μg/ml as a counterstain.

The slides were examined with an FITC/Texas red double-band pass filterset (Omega Optical on a Zeiss Axioskap.

PCR Method of Kohler et al

The method of Kohler et al. (Leukemia 4, 8 (1990)] for bcr-abl PCR onCML-2, CML-4, and CML-5. The oligonucleotide primers used were asfollows: ablX3 antisense downstream 5′-TTT CTC CAG ACT GTT GAC TGG-3′;ablX2 sense upstream 5′-CCT TCA GCG GCC AGT AGC AT-3′; CML bcr upstream5′-ACA GCA TTC CGC TGA CCA TC-3′; CML abl antisense detection 5′-TAT GCTTAG AGT GTT ATC TCC ACT-3′.

PCR Method of Hogewisch

Method used for bcr-abl PCR by Hogewisch-Becker et al. [J. Biol. Chem.Suppl. 188, 289 (1989)] on cases CML-3a, CML-3b, and CML-6. Theoligonucleotide primers used were as follows: sense primer (upstream ofbcr) 5′-AGG GTG CAC AGC CGC AAC GGC-3′; antisense primer (abl)5′-CGC TTCACT CAG ACC CTG AGG-5′; probe for the identification of bcr3/ab12junction sequence 5′-GAA GGG CTT TTG AAC TCT G-3′; probe for theidentification of bcr2abl2 junction sequence 5′-GAA GCG CTT CTT CCTTAT-3′. Exon 3 of bcr is joined to abl exon 2 if a 314-bp fragment isamplified. Exon 2 of bcr is joined to abl exon 2 if a 239-bp fragment isamplified.

Southern Blot

Southern blot analysis on case CML-4 showed a rearranged Bg1 II bandusing an OSI Transprobe-1 Kit (oncogene Science catalog no. TP88).

Construction of a PEM Library (Maniatis, 1982)

The PEM library was made from human placental DNA. This is considered tobe “normal” DNA. The DNA was prepared by partial MbaI digestion, sizeselection, and ligation to the vector EMBL3 at the BAMHI site.

The vector inserts in the 15-20 kb size range. The insert can be excisedwith SalI. The Bam site is usually lost due to ligation to MbaI.

The insert-containing phage has no EcoRI sites, whereas the wild-typephage has EcoRI sites.

Phage may be grown on NM539 media. 10-12 plates of 50,000 clones eachare generally screened.

The library has been amplified. This generally leads to a titer drop ofabout 10-fold.

Protocol for Construction of a Cosmid Library (Maniatis, 1982)

A cosmid library was plated on amphicillin plates and amplified onchloromphenical.

Phage were grown on filters in 20 plates. The titre was about 8×10screened colonies per plate. Normally 2-4 of a few genome equivalentswere obtained.

Replica plating was performed, 2 sets of each plates were screened witha probe.

To prepare a probe, generally an insert was cut out of a plasmid so thatthe plasmid sequences did not cross hybridize.

An ECQ fragment 228-1-2 (a 3′ probe) was made by this method. It wasthen nick translated. An oligonucleotide may also be preparedsynthetically. The duplicate filters were treated to fix and denaturethem by techniques known to those of skill in the art.

Each filter was treated on a column with

0.5M NaOH

1.5M NaCl

and moved to

1M TRIS ph 7.5

then into

0.5 TRIS 7.5

1.5M NaCl

Filters were blotted with 13M filter paper and dried at 68° C.

Nitrocellulase paper not nylon was preferred

A pre-hybridization mix was used to screen the library at 68° C.

The label was 7×10⁶ CPM p³² for the probe. Hybridization was performedat 42° C.

Washed in 2×SSC 5% SDS

(68%) for 2-3 hours

The filters were exposed until colonies were visible (˜3 days)

Positive colonies were scored.

Colony re-purification was performed by rescreening in a similarprocedure to the above.

Phage

2 clones were isolated from a phage library (human genome library ofanonymous human placenta).

EMBO 1 partial digestion of DNA was performed.

DNA size was selected on a sucrose gradient to be in the 15-20 kbaverage size.

The fragments were ligated into the BAM HI site of the EMBO 3 vector(Stratogene or Promega).

The PEM12 clone was plated and the library screened 50,000 clones with 2probes. The result was 2 clones.

These were screened with a BCR exon probe.

The insert was ˜16⁶ kb.

Colony Biotechnology Systems, NEM Research Products Plaque Screen CatNEF 978/978A 978X/978Y

U.S. Pat. No. 4,455,370 du Pont de Nemours.

References

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

Anastasi, J., Thangavelu, M., Vardiman, J. W., Hooberman, A. L., Bian,M. L., Larson, R. A., and LeBeau, M. M. (1991), Interphase cytogeneticanalysis detects minimal residual disease in a case of acutelymphoblastic leukemia and resolves the question of origin of relapseafter allogeneic bone marrow transplantation. Blood 77:1087-1091.

Arnoldus, E. P. J., Wiegant, J., Noordermeer, I. A., et al. (1990),Detection of the Philadelphia chromosome in interphase nuclei. CytogenetCell. Genes. 54:108-111.

Bartram, et al. (1987), Blut, 55:505-511.

Benn, et al. (1987), Cancer Genes. Cytogenet, 29:1-7.

Blennerhassett, et al. (1988), Leukemia, 2:648-57.

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4 21 base pairs nucleic acid single linear DNA 1 AGGGTGCACA GCCGCAACGG C21 21 base pairs nucleic acid single linear DNA 2 CGCTTCACTC AGACCCTGAGG 21 19 base pairs nucleic acid single linear DNA 3 GAAGGGCTTT TGAACTCTG19 18 base pairs nucleic acid single linear DNA 4 GAAGCGCTTC TTCCTTAT 18

What is claimed is:
 1. A method of detecting in a sample of chromosomesa chromosomal translocation associated with cancer, wherein thetranslocation results in at least one fusion chromosome, said methodcomprising: (a) preparing at least a first, a second and a third genomicnucleic acid probe, said first and second probe designed to hybridize toa fusion chromosome on one side of the fusion breakpoint region, but atdifferent locations and said third probe is designed to hybridize to achromosome on the other side of the breakpoint fusion region, whereinsaid first, second and third probes are distinctly labeled so that eachis distinguishable; (b) contacting the labeled probes in pairs whereinthe third probe is a member of each pair, to the sample containing thechromosomes under conditions of appropriate stringency to allow specifichybridization of the probes to complementary sequences within the DNA ofsaid chromosomes; and (c) detecting the presence of label from the threeprobes hybridized to the chromosomes; and (d) inferring the presence ofthe translocation from the label pattern.
 2. The method of detecting achromosomal translocation of claim 1, wherein the probes arefluorescently labeled.
 3. The method of detecting a chromodomaltranslocation of claim 2, wherin the florescent lables aredistinguishable under a microscope as different colors.
 4. The method ofdetecting a chromosomal translocation of claim 3, wherin the florescentlables are attached to digoxigenin-11-duTP and biotin-11-duTP.
 5. Themethod of detecting a chromosomal translocation of claim 1, wherin thefirst and second probes hybridize to sequences that are at leastapproximately 800 kb apart in a fusion chromosome from the sequence towhich the third probe hybridizes.
 6. The method of detecting achromosomal translocation of claim 1, wherein the probes hybridize withchromosomal DNA in situ in cells.
 7. The method of detecting achromosomal translocation of claim 6, wherein the cells comprise thosein interphase of mitotic division.
 8. The method of detecting achromosomal translocation of claim 7, wherein the probes afterhybridization are juxtaposed as doublets if a chromosomal translocationis present.
 9. The method of detecting a chromosomal translocation ofclaim 8, wherein the translocation is formed by breakpoints which occuron the long arms of human chromosomes 9 and
 22. 10. The method ofdetecting a chromosomal translocation of claim 9, wherein thetranslocation breakpoints are further defined as occurring at locationst(9;22)(q11;q34).
 11. The method of detecting a chromosomaltranslocation of claim 10, wherein the translocation breakpoints arefurther defined to occur in the BCR and ABL genes respectively, and afusion gene is formed comprising portions of the BCR and ABL genes. 12.The method of detecting a chromosomal translocation of claim 6 whereinthe cells comprise a sample of human tissue.
 13. The method of detectinga chromosomal translocation of claim 12, wherein the human tissue samplecomprises peripheral blood.
 14. The method of detecting a chromosomaltranslocation of claim 12, wherein the human tissue sample comprisesbone marrow.
 15. The method of detecting a chromosomal translocation ofclaim 6, wherein the cells comprise a sample of cultured cells.
 16. Themethod of detecting a chromosomal translocation of claim 1, wherein saidprobes comprise the inserts of MSB-1, PEM12 and c-H-abl, and said probesare contacted to chromosomes in pairs.
 17. A method of dectecting achromosomal translocation resulting in a fusion gene between BCR and ABLgenes associated with cancer, said method comprising: (a) preparing atleast three nucleic acid probes, each labeled with distinguishablelabel, wherein a first of said probes hybridizies to the BCR gene sideof the translocation, a second of said probes hybridizes to a differentpart of the BCR gene than the part to which the first probe hybridized,and the third probe hybridizes to part of the ABL gene; (b) contactingsaid probes in pairs, wherein the third probe is a member of each pairto chromosomes under conditions of appropriate stringency to allowspecific hybridization of the probes to complementary DNA sequences inthe chromosomes; (c) detecting the presence of hybridized probes; and(d) identifying said chromosomal translocation by the labeling patternof the probes relative to the chromosomes; wherein said resulting fusiongene encodes either of two proteins designated as p190 and p210.
 18. Themethod of detecting a chromosomal translocation of claim 17, wherein thefusion gene encodes a protein designated as p190.
 19. The method ofclaim 12, wherein the presence of said fusion gene is diagnostic foracute lymphocytic leukemia (ALL).
 20. The method of detecting achromosomal translocation of claim 17, wherein the fusion gene encodes aprotein designated as p210.
 21. The method of detecting a chromosomaltranslocation of claim 12, wherein the probes consist of those selectedfrom PEM12, c-H-abl and MSB-1.
 22. The method of detecting chromosomaltranslocations of claim 16, wherein a first pair comprises the insertsof MSB-1 and c-H-abl, and a second pair comprises the inserts of PEM12and c-H-abl.
 23. The method of detecting a chromosomal translocation ofclaim 22, wherein the first pair of probes detects a fusion geneencoding a protein designated as p190 and the second pair of probesdetects a fusion gene encoding a protein designated as p210.
 24. Themethod of detecting a chromosomal translocation of claim 23, wherein thepresence of said chromosomal translocation is diagnostic for ALL orchronic myelogenous leukemia (CML).
 25. The genetic probe of claim 22,wherein the probe comprises at least part of the nucleic acid sequenceof the insert of MSB-1.
 26. The genetic probe of claim 23, wherein theprobe comprises at least part of the nucleic acid sequence of the insertof c-H-abl.
 27. A method of detecting a structural chromosomaltranslocation comprising: (a) contacting probes prepared from theinserts of MSB-1 and c-H-abl with chromosomal DNA from a human subject;(b) contacting probes prepared from the inserts of PEM12 and c-H-ablwith chromosomal DNA from the same subject; (c) detecting the presenceof probe molecules hybridized to complementary sequences within thechromosomal DNA; and (d) identifying the specific chromosomaltranslocation present in the subject by the pattern of hybridization ofthe subject's chromosomal DNA with said probes.
 28. The method ofdetecting a chromosomal translocation of claim 27 wherein the probes arefluorescently labeled.
 29. The method of detecting a chromosomaltranslocation of claim 28 wherein each probe label is distinct from eachother.
 30. The method of detecting a chromosomal translocation of claim29 wherein the fluorescent labels are distinguishable under a microscopeas different colors.
 31. The method of detecting a chromosomaltranslocation of claim 27 wherein the probes after hybridization arejuxtaposed as doublets if a chromosomal translocation is present. 32.The method of detecting a chromosomal translocation of claim 27 whereinthe presence of said chromosomal translocation is diagnostic for ALL.33. The method of detecting a chromosomal translocation of claim 27wherein the presence of said chromosomal translocation is prognostic forALL.
 34. The method of detecting a chromosomal translocation of claim 27wherein the presence of said chromosomal translocation is diagnostic forchronic myelogenous leukemia (CML).
 35. The method of detecting achromosomal translocation of claim 27 wherin the presence of saidchromosomal translocation is prognostic for CML.
 36. A method ofdetecting a chromosomal translocation resulting in a fusion gene betweenBCR and ABL, said fusion gene associated with cancer, said methodcomprising: (a) preparing at least two nucleic acid probes, each labeledwith a distinguishable label, wherein a first of said probes hybridizesto the BCR gene side of the translocation, and the third probehybridizes to a part of the ABL gene; (b) contacting said probes tochromosomes under conditions of appropriate stringency to allow specifichybridization of the probes to complementary DNA sequences in thechromosomes; (c) detecting the presence of hybridized probes; and (d)identifying the chromosomal translocation by the labeling pattern of theprobes relative to the chromosomes; wherein said translocation resultsin a fusion gene that encodes either of two proteins designated as p190and p210.